JPH08120000A - Anti-human vitronectin/thrombin/antithrombin iii complex monoclonal antibody, hybridoma and immunoassay - Google Patents

Abstract

PURPOSE: To obtain the subject new antibody for diagnosing blood coagulation blood coagulation sthenia, accentuation, specifically reacting with vitronectin/ thrombin/antithrombin III complex (VTAT), not reacting with each liberated material, capable of measuring VTAT in high sensitivity. CONSTITUTION: Vitronectin is reacted with thrombin and antithrombin III in 1M tris hydrochloric acid buffer solution (pH7.5) containing 0.9% NaCl at 37 deg.C for 30 minutes and purified by molecular sieve chromatography to obtain VTAT. Then VTAT and an adjuvant are administered to the abdominal cavity of a mouse and the mouse is immunized. After the final immunization, splenic cells are collected, fused with mouse myeloma cells and selectively cultured in a HAT medium to give a hybridoma. Then the hybridoma is cloned by a limiting dilution method to establish an antibody forming strain. The antibody forming strain is cultured to give the VTAT monoclonal antibody which specifically reacts with VTAT but not with liberated vitronectin, thrombin and antithrombin III.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an anti-vitronectin / thrombin / antithrombin III complex monoclonal antibody, a hybridoma secreting said monoclonal antibody, and an immunological immunoassay of vitronectin / thrombin / antithrombin III complex using said monoclonal antibody. Regarding measurement method.

[0002]

2. Description of the Related Art In the clinical field, measuring thrombin in blood is important for knowing hypercoagulable states such as disseminated intravascular coagulation and thrombosis. However, since it is not possible to measure thrombin itself, instead of thrombin, antithrombin III in blood (hereinafter referred to as AT
(Also called III) and thrombin
The antithrombin III complex (hereinafter also referred to as TAT) is measured, and the amount of thrombin is estimated from the amount to be used as an index of coagulation activation of blood. However, TAT rapidly binds to vitronectin in blood, and the vitronectin-thrombin-antithrombin III complex (VT
AT) is formed and metabolized. Therefore, it is not clear in a strict sense whether it is appropriate to use TAT as an index. It is also considered more appropriate to use VTAT as an index.

In practice, the vitronectin / thrombin / antithrombin III complex (VTAT) was also measured by the conventional method. In the measurement method, an anti-antithrombin III monoclonal antibody is immobilized on a solid phase, and a VTAT complex bound to a solid phase antibody obtained by contacting a body fluid sample such as human plasma with an enzyme-labeled anti-vitronectin monoclonal antibody. It was carried out by acting.

[0004]

However, in this method, VTAT coexisting in a body fluid sample such as human plasma is used.
Excessive (about 100,000 times) antithrombin III compared to
It is impossible to escape the interference of the VTAT
Cannot be measured (JP-A-2-66458). Also, TAT cannot be measured. Therefore, it is an object of the present invention to provide a novel monoclonal antibody, which is capable of accurately producing V without the interference of antithrombin III.
It is to provide a means for measuring TAT.

[0005]

That is, the present invention specifically reacts with vitronectin / thrombin / antithrombin III complex (VTAT), but free vitronectin, free thrombin and free antithrombin III.
Does not react with (ATIII), preferably thrombin.
It relates to a monoclonal antibody characterized by not reacting with antithrombin III complex (TAT). Furthermore,
The present invention also relates to a hybridoma characterized by secreting the above monoclonal antibody, and a method for immunologically measuring vitronectin / thrombin / antithrombin III complex (VTAT) characterized by using the above monoclonal antibody. . Furthermore, the present invention provides a thrombin / antithrombin together with the above monoclonal antibody.
A monoclonal antibody that specifically reacts with the III complex (TAT), but does not react with free thrombin and antithrombin III, was used together with vitronectin / thrombin / antithrombin III complex (VTAT) to form the thrombin / antithrombin III complex. It also relates to a method for immunologically measuring the body (TAT).

The present invention will be described in detail below. The anti-VTAT monoclonal antibody of the present invention is a novel hybridoma (preferably mouse hybridoma) of the present invention.
Can be produced by culturing in the medium or in the abdominal cavity of a mammal (particularly mouse). The hybridoma according to the present invention generally comprises spleen cells and mouse myeloma cells of a mouse immunized with VTAT as Koh
Basic Method for Cell Fusion of Ler and Milistin [Nature, 256, 495 (1975)
See]. Details will be described in the following examples.

The medium for culturing the above hybridoma may be any medium suitable for culturing the hybridoma, and is preferably Dulbecco's modified essential essential medium (Dulbecco's modified Ee).
agle's minimum essential
(hereinafter referred to as DME), fetal bovine serum,
A medium containing L-glutamine, L-pyruvate and antibiotics (penicillin G and streptomycin) is used.

The anti-VTAT monoclonal antibody described above can be separated and purified from the thus-produced culture medium or ascites of mouse by a method generally used for protein isolation and purification. Examples of such methods include salting out with ammonium sulfate, ion exchange column chromatography using ion exchange cellulose, molecular section column chromatography using molecular section gel, affinity column chromatography using protein A-bound polysaccharide, dialysis,
Freeze drying etc.

The anti-VTA of the present invention thus obtained
The T monoclonal antibody has the ability to bind only to VTAT without binding to free vitronectin, free ATIII and free thrombin, and preferably does not further react with TAT, and is useful as a reagent for immunoassay of VTAT. Is. The anti-VTAT monoclonal antibody of the present invention (for example, VAT-1 and VAT-obtained in Examples described later)
2) can be used as a reagent in enzyme immunoassay (EIA) or luminescence immunoassay (LIA). As an example of enzyme immunoassay method, one-step method using microtiter or plastic tube
A sandwich enzyme immunoassay can be mentioned. A specific example of this enzyme-linked immunosorbent assay is as shown in Example 7 below, but generally, two types of anti-VTAT monoclonal antibodies that recognize different determinants of VTAT are used. Pre-set the microtiter plate holes (wells) or plastic tubes
It is sensitized with a different type of antibody (eg VAT-1), and then in this hole or in a plastic tube, the analyte containing VTAT and another type of enzyme-labeled antibody (eg VA)
The solution of T-2) is added, and the solution is allowed to stand for about 30 minutes and then cleaned, an enzyme substrate solution is added, and an enzyme reaction is performed for about 30 minutes. After completion of the reaction, it can be performed by quantifying the amount of VTAT in the subject by a colorimetric method or the like.

When the luminescent immunoassay (LIA) is used, one kind of the anti-VTAT monoclonal antibody of the present invention (for example, VAT-2) is sensitized to a carrier such as polystyrene beads and then sensitized. An analyte containing VTAT is contacted with the carrier, and the carrier is washed if necessary, and then a solution of another type of antibody (for example, VAT-1) labeled with a luminescent substance is added to the reaction to cause reaction. This can be performed by adjusting the atmosphere to the conditions for emitting light and measuring the amount of emitted light with a Lumiphotometer to quantify the amount of VTAT in the subject. Luminol, an acridinium derivative, or the like can be used as the light-emitting substance.

When the monoclonal antibody of the present invention is used in a latex agglutination immunoassay, the latex sensitized with the monoclonal antibody of the present invention is contacted with a test sample,
By measuring the presence / absence of aggregation associated therewith, V in the subject is measured.
TAT can be quantified. The latex used is not particularly limited, and polystyrene latex particles can be used, for example.

The monoclonal antibody of the present invention is used in combination with a monoclonal antibody that specifically reacts with the thrombin / antithrombin III complex, but does not react with free thrombin or free antithrombin III.
Also, TAT can be measured immunologically. As the anti-TAT monoclonal antibody, for example, the monoclonal antibody described in JP-A-62-138187, particularly AT-1 can be used.

When simultaneously measuring VTAT and TAT, the enzyme immunoassay, chemiluminescence immunoassay or latex agglutination immunoassay can be used as described above. For example, in the case of the latex agglomeration method, the VA of the present invention
T-1 and / or VAT-2 and the above-mentioned AT-1 are separately sensitized to latex particles separately, or a mixture of the above-mentioned monoclonal antibodies is sensitized to latex particles to obtain a sample in a subject. Contact with VTAT of
The presence / absence of aggregation associated therewith may be measured. In the case of enzyme immunoassay or luminescence immunoassay, the TAT-1 and / or VAT of the present invention can be used as a carrier for plates and beads.
-2 and AT-1 described above are immobilized, and the same operation as described above may be performed using a different type of monoclonal antibody or anti-antithrombin III antibody (polyclonal antibody) of the present invention labeled with an enzyme or a luminescent substance. . In the various measurement methods according to the present invention, the test sample is not particularly limited as long as it is a sample that may contain VTAT and TAT in some cases.
Particularly, blood, serum, plasma and the like can be mentioned.

[0014]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 The VTAT complex was transformed into E. R. The method of Podak et al. [Journal of Biological Chemistry, Vol. 261,
7387-7392 (1986)].
That is, 800 μg of vitronectin and 40 of thrombin
0 μg and 800 μg of antithrombin III in 0.1 M Tris-HCl buffer (pH: 0.9%)
7.5) Add to 1 ml and react at 37 ° C for 30 minutes,
The VTAT complex was prepared. Furthermore, Sepha Acrylic S
-VTAT (Pharmer, Sweden) purified by molecular sieve chromatography and purified VTAT
(A280 nm = 1.0 VTAT) 1 ml was obtained. The purified VTAT thus obtained was used as an immunogen and an antigen for ELISA for selecting anti-VTAT antibody-producing hybridomas in the following examples.

Example 2 (a) Preparation of immunized spleen cells: The VTAT immunogenic solution prepared in Example 1 (A280 nm = 0.1) was mixed with an equal volume of Freund's complete adjuvant until emulsified. Immunization was performed by intraperitoneally administering 200 μl of the mixed solution to the mouse (first immunization). After 30 days,
The mice were intraperitoneally administered by the same method as described above (second immunization). 21 days after the second immunization, VTAT
The immunogen solution (A280 nm = 0.1) was diluted with an equal volume of physiological saline, and 200 μl of the diluted solution was intravenously administered to the mouse (final immunization). 3 days after the final immunization,
Spleen cells were removed from the mice and used for cell fusion.

(B) Cell fusion: The above-mentioned spleen that has been aseptically removed is used in DME medium 5 containing 10 to 15% fetal bovine serum.
It was put in a petri dish containing ml. Next, spleen 10
The spleen cells were allowed to flow out by refluxing with about 15 ml of DME medium containing 15% fetal bovine serum, and this spleen cell suspension was passed through a nylon mesh. 50 ml of the splenocytes were collected in a centrifuge tube and centrifuged (500 xg, 10 minutes). The pellet thus obtained was hemolyzed (155 mM-N
H ₄ Cl, 10 mM-KHCO ₃ , 1 mM-Na ₂ ED
5 ml of TA, pH 7.0) was added and suspended. 0 ° C
When left for 5 to 10 minutes, the red blood cells in the suspension were destroyed. DME containing 15 to 15% fetal bovine serum
The medium was added and then centrifuged. The cell pellet thus obtained was washed with DME medium by centrifugation, and the number of living splenocytes was measured.

On the other hand, mouse myeloma cells (myeloma cells) SP2 / O-Ag14 about 2 × 10 5 pre-cultured.
About 1 × 10 ⁸ of the above spleen cells were added to ⁷ cells, mixed well in DME medium, and centrifuged (500 × g, 10 minutes). Aspirate the supernatant, loosen the pellet well,
0.5 ml of 40% polyethylene glycol 4000 solution kept warm at 38 ° C. was added dropwise, and the centrifugation tube was gently rotated by hand for 1 minute to mix the polyethylene glycol solution with the cell pellet. Then 38
Every 30 seconds, 1 ml of the DME medium kept at 0 ° C. was added, and the tube was gently rotated. After repeating this operation 10 times, DME containing 10 to 15% fetal calf serum
20 ml of medium was added, and centrifugation (500 × g, 10 minutes) was performed. After removing the supernatant, 10
HAT medium containing ~ 15% fetal bovine serum (aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ^{−5 in} DME medium)
M and hypoxanthine (1 × 10 ⁻⁴ M were added), and the cells were washed twice by centrifugation and then suspended in 40 ml of the HAT medium. 200 μl of this cell suspension was dispensed into each well of a 96-well cell culture plate,
Culturing was started at 0 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. During the culturing, the medium in each well was adjusted to about 10 at intervals of 2 to 3 days.
Hybridomas that grow in HAT medium were selected by removing 100 μl of the above HAT medium except 0 μl. HT medium containing 10 to 15% fetal bovine serum from about the 8th day (thymidine 1.6 × 10 ⁻⁵ M in DME medium,
Hypoxanthine (1 × 10 ⁻⁴ M was added) to observe the growth of hybridomas, and about 10 days later, anti-VT was measured by the ELISA method described below.
The AT antibody producing hybridoma was screened.

(C) Establishment of hybridoma The presence or absence of produced antibody in the hybridoma culture supernatant is determined by ELSI.
It was measured by Method A. To each well of a 96-well ELISA plate (Immulon II; Nippon Dynatech Co., Ltd.), the above-mentioned purified VTAT solution (A280 nm =
0.05, diluted with physiological saline) was dispensed in 50 μl aliquots and left at 25 ° C. for 2 hours. Next, 0.05% T
ween20 (registered trademark of ICI) -3 with saline
After washing twice, add 50 μl of culture supernatant to each well, and
The reaction was carried out at 5 ° C for 1 hour. Next, 0.05% Tween
50 μl of peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold with 20-saline was added to each well. After completion of the reaction, 0.05% Tween
Wash each well 3 times with 20-saline, 0.5 mM
Aminoantipyrine and 10 mM phenol and 0.005
Add 250 μl of a solution containing 100% hydrogen peroxide to each well and react at 25 ° C. for 30 minutes.
The absorbance at was measured. As a result, antibody production was observed in 23 of 192 wells.

The anti-VTAT antibody in the culture supernatant confirmed by the above-mentioned ELISA method is vitronectin, ATII.
Whether or not it reacts with I 2, thrombin and TAT was measured in the same manner as above using a 96-well ELISA plate sensitized with vitronectin, ATIII, thrombin and TAT. As a result, among the 15-well culture supernatants that reacted with VTAT, 8 wells of the culture supernatant were TAT and AT.
III and 5 well culture supernatants with vitronectin,
Reacted On the other hand, it did not react with thrombin at all.
The hybridomas in 2 wells which did not react with vitronectin, ATIII, thrombin and TAT but specifically react with VTAT only were transferred to a 24-well plate, and 1
The cells were cultured in HT medium containing 0 to 15% fetal bovine serum for 4 to 5 days. After that, the "anti-VTA" was again determined by the ELISA method.
After confirming the production of "T-specific antibody" (hereinafter simply referred to as anti-VTAT antibody), cloning was performed by the limiting dilution method. In the limiting dilution method, a cell suspension diluted with HT medium to 5 hybridomas / ml was prepared in advance with normal B
Peritoneal cells of ALB / C mouse are 2 × 10 5 per well
10 in each well of a 96-well plate in which ⁴ tubes are dispensed
Aliquots of 0 μl were dispensed. About 10 days later, a hybridoma clone producing an anti-VTAT antibody was screened by the ELISA method. As a result, 20 to 40 antibody-producing clones were obtained for each hybridoma.
From these clones, a clone with good proliferation, high antibody secretion ability, and stable, was selected and recloned in the same manner as described above to obtain "anti-VTAT-specific antibody".
Production hybridomas VAT-1 and VAT-2 were established. These hybridomas VAT-1 and VAT-
The antibody secreted from 2 did not react with vitronectin-thrombin complex (VT).

Example 3: Production of monoclonal antibody (a) In vitro method Mouse hybridomas VAT-1 and VAT-2 were each incubated at 37 ° C. in DME medium containing 15% fetal calf serum.
The cells were cultured in a 5% carbon dioxide atmosphere for 72 to 96 hours. After centrifuging the culture (10,000 × g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture was stirred under ice cooling for 30 minutes, then left for 60 minutes and centrifuged (10000 xg, 10
After that, the resulting precipitate was dissolved in a small amount of 10 mM phosphate buffer (pH 8.0) and dialyzed against 1000 times volume of 10 mM phosphate buffer. This was loaded onto a column of DEAE-cellulose already equilibrated with 10 mM phosphate buffer. Elution of the monoclonal antibody was performed with 10 mM phosphate buffer (pH 8.0) and 0.2 M NaCl 1
It was performed by a concentration gradient method between 0 mM phosphate buffer (pH 8.0). The eluted monoclonal antibody was concentrated by ultrafiltration and dialyzed against 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG-Sepharose 4B to remove bovine serum IgG. Next, the passing solution was treated with 0.1 M phosphate buffer (pH 8.
0) Equilibrated protein A-Sepharose 4B column. The column was eluted with a pH 3.5 buffer to obtain a purified anti-VTAT antibody VAT-1 solution.
Similarly, a solution of VAT-2 was obtained.

(B) In vivo method 0.5 ml of pristane (2,6,1,14-tetramethylpentadecane) was intraperitoneally administered to BALB / C mice aged 10 to 12 weeks, and then 14 to 20. Hybridoma VAT grown in vitro in mouse peritoneal cavity on day
-1, or VAT-2, was inoculated to each mouse at 2 × 10 ⁶ cells. About 10 to 15 ml of ascites was obtained from one mouse for each hybridoma. The antibody concentration was 2-10 mg / ml. Purification of monoclonal antibody in ascites (however, goat anti-bovine serum IgG
-Excluding the operation of passing the column of Sepharose 4B) was performed in the same manner as the above in vitro purification method.

Example 4: Immunoglobulin of monoclonal antibody
Identification of Bryn class and specificity Anti-VTAT monoclonal antibodies VAT-1 and VAT-
Identification of the immunoglobulin class and specificity of 2 was performed by the octelony immunodiffusion method and the enzyme immunoassay method [the same method as the ELISA method described in Example 2 (c) above]. The results are shown in Table 1 and FIG. 1 (VA
T-1) and FIG. 2 (VAT-2). 1 and 2
, T is thrombin, A is antithrombin III
, VT is vitronectin-thrombin complex, TAT
Is the thrombin-antithrombin III complex, and V
TAT is a vitronectin / thrombin / antithrombin III complex.

[0023]

[Table 1] Immunoglobulin class monoclonal antibody Immunoglobulin class VAT-1 IgG ₁ VAT-2 IgG ₁

Example 5: Latex slide aggregation immunoassay
The amount method anti-VTAT monoclonal antibody according to the latex (manufactured by Japan Synthetic Rubber Co., Ltd., 0.497μm) sensitization was carried out in the following operations. Solution 1 in which the concentration of each of the two types of monoclonal antibodies VAT-1 and VAT-2 is 0.3 mg / ml
Latex was added to 0 ml so that the latex concentration was 1% (w / v), and the mixture was vigorously stirred at 25 ° C. for 1 hour.
Next, 0.2m of bovine albumin solution (10mg / ml)
1 was added, and the mixture was vigorously stirred at 25 ° C. for 30 minutes and then centrifuged (20,000 × g, 30 minutes). Water 5
It was suspended in 0 ml and used as latex VTAT • L-1 sensitized with anti-VTAT monoclonal antibody in the following latex slide aggregation immunoassay. Next, monoclonal antibody VAT-1 and Example 3 of JP-A No. 62-138187.
A sensitized latex was prepared in the same manner as described above using the combination with the monoclonal antibody AT-1 which specifically reacts with TAT described in 1.
It was used as AT.L-2 in the following latex slide aggregation immunoassay.

25 μl of the sensitized latex (0.2%) was added to each well of the slide aggregation plate, and then 25 μl of the test substance in a 2-fold dilution series with physiological saline was added. This slide aggregation plate was rotated at 60 rpm for 3 minutes, and the presence or absence of aggregation in each well was measured. The amount of VTAT in the test sample was determined from the presence or absence of the latex agglutination reaction due to the calibration complex in the 2-fold dilution series that was simultaneously measured. Table 2 shows the results.

[0026]

[Table 2] VTAT concentration (ng / ml) 160 80 40 20 10 5.0 2.5 1.25 VTAT ・ L-1 ＋＋＋ －－－－－ VTAT ・ L-2 ＋＋＋＋＋＋＋＋＋ In Table 2, + means aggregation , And-represent no aggregation.

Example 6: One-step sandwich
Enzyme-linked immunosorbent assay Antibody solution containing anti-VTAT monoclonal antibody VAT-2 in 20 mM carbonate buffer at a concentration of 10 μg / ml, and separately from anti-VTAT monoclonal antibody VAT-2 and anti-TAT monoclonal antibody used in Example 6 AT-
An antibody solution containing 1 of 1 in 20 mM carbonate buffer at a concentration of 5 μg / ml was prepared, and 100 μl was added to each 96-well flat bottom polystyrene microtiter plate.
It was put in an amount of 1 and left still at 25 ° C. for 30 minutes. The plate was washed 3 times with saline containing 0.05% Tween-20. In a well of the antibody-sensitized plate thus obtained, 50 μl of a test substance, 20 mM phosphate buffer containing peroxidase-labeled anti-human antithrombin III antibody (Rabbit, Dako: Denmark), 0.15 M-NaCl, and 2% bovine albumin. (PH 8.0) 100 μl
Was added. After standing at 25 ° C. for 30 minutes, the plate was washed with 0.
It was washed three times with a physiological saline solution containing 05% Tween-20. Then, the enzyme substrate solution (10 mM phenol, 20 m
200 μl of a solution containing M-4-aminoantipyrine and 0.005% hydrogen peroxide was added to each well of the plate. After reacting at 25 ° C. for 30 minutes, the absorbance at 490 nm of each well was measured by MP-590 type MacroElisa Mini Reader (manufactured by Dynatech), and the amount of VTAT in the test substance was measured at the same time. It was determined from a calibration curve drawn from the absorbance of the body at 490 nm. The results are shown in Fig. 3. In FIG. 3, A is the monoclonal antibody VAT-2 of the present invention and JP-A-62-1.
The results are obtained by immobilizing two types of TAT-specific monoclonal antibody AT-1 described in Example 3 of 38187 publication, and B is the monoclonal antibody VAT of the present invention.
It is a result when -2 is immobilized.

[0028]

As described above, VTAT can be quantified with high sensitivity by using the anti-VTAT monoclonal antibody of the present invention. In addition, by combining the anti-VTAT monoclonal antibody of the present invention with the anti-TAT monoclonal antibody, both TATs that do not bind to vitronectin can be measured in a sample, and both can be integrated and integrated with thrombin. It has the advantage of being able to grasp.

[Brief description of drawings]

FIG. 1 Anti-VTAT monoclonal antibody V according to the invention
Vitronectin thrombin of AT-1, ATIII, V
It is a graph showing the result of having measured the binding force with T, TAT, and VTAT by the enzyme immunoassay method using 96 well microtiter plate.

FIG. 2 Anti-VTAT monoclonal antibody V according to the invention
AT-2 vitronectin thrombin, ATIII, V
It is a graph showing the result of having measured the binding force with T, TAT, and VTAT by the enzyme immunoassay method using 96 well microtiter plate.

FIG. 3 is a graph showing a calibration curve of a VTAT sandwich EIA method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 5/10 15/02 C12P 21/08 9358-4B G01N 33/53 L 33/533 33/577 B // (C12P 21/08 C12R 1:91) (72) Inventor Yumiko Ito 1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd.

Claims (5)

[Claims] 1. Free vitronectin, free thrombin and free antithrombin II, which react specifically with the vitronectin / thrombin / antithrombin III complex.
A monoclonal antibody characterized by not reacting with I. 2. Free vitronectin, free thrombin and free antithrombin II which react specifically with the vitronectin / thrombin / antithrombin III complex.
A hybridoma characterized by secreting a monoclonal antibody that does not react with I. 3. Free vitronectin, free thrombin and free antithrombin II, which react specifically with vitronectin / thrombin / antithrombin III complex.
An immunological assay method for vitronectin / thrombin / antithrombin III complex, which comprises using a monoclonal antibody which does not react with I. 4. The thrombin-antithrombin III complex is simultaneously quantified by using together a monoclonal antibody that specifically reacts with the thrombin-antithrombin III complex but does not react with free thrombin and free antithrombin III. The method according to 3. 5. The method according to claim 3, wherein the immunoassay method is an enzyme immunoassay method, a chemiluminescence immunoassay method, or a latex agglutination immunoassay method.